The present invention relates to test devices and methods for the determination of analytes which may be present in liquids.
The quantification of chemical and biological components in aqueous solutions such as whole blood, plasma, serum and urine is important for the timely and correct diagnosis of various diseases, as well as for monitoring the progress of the medical treatment of diseases. In many cases the analytes being measured are present in only tiny amounts and are often mixed with much larger amounts of irrelevant or interfering components. Some components such as red blood cells prevent the analysis of the sample if they are present. Also problematic are the reagents and indicators used to detect and measure the analytes. These reagents are often highly colored and closely resemble the reaction products in terms of their absorbance spectra. In addition, the measurement of analytes often requires multiple, incompatible reagents that must be stored separately and added sequentially. Any of these factors may complicate the detection and quantification of analytes in fluid samples.
These problems and issues have been addressed in a variety of ways. Analytical methods used can be divided into two broad categories of assay formats: liquid chemistry formats and dry chemistry formats. Liquid chemistry systems require that sample and liquid reagents be dispensed into reaction chambers in a timed, sequential order. Samples must often be diluted with special buffers to reduce or eliminate interfering compounds and are then added to reagents designed to react with specific analytes. In some cases, multiple reagents must be premixed immediately before use due to stability problems. In other cases additional reagents may be needed to provide color-producing, readable reactions. The results may be obtained by measuring the absorption of light by the fluid sample. Reactions involving decreases in reaction color or minor differences in color changes may further require separate tubes of reagents to standardize the results or serve as controls.
Dry chemistry systems utilize reagents dried onto absorbent surfaces. Most commercially available products have multiple layers of reactants sandwiched together. Some are arranged vertically and some combine vertical and horizontal arrangements. In all cases, dry chemistry systems using chromogenic reactions rely on measuring light reflected off of either the top or bottom surface of the final reagent pad. The assaying of whole blood presents additional problems since it requires a separate method for separating red blood cells from the sample such as centrifugation or the use of blood separating filter(s) which separate the plasma for analysis. The essence of dry chemistry analysis is to contain a liquid reaction so that colored reaction products can be visualized. This is done with gels and polymers manufactured by, for example, Kodac/Reflotron on fibrous paper-like materials manufactured by, for example, Miles/Ames. In all cases the reaction must be observed among a mixture of sample, diluant, reactants and product which can result in difficulties distinguishing product from non-product. In addition, since all or part of the original reactant is consumed, it can be impossible to reference back to the starting material, such as to establish a reagent baseline. In contrast, the present invention retains all components for further evaluations.
In methods that require precise reaction timing, such as those requiring rapid reactions or measuring a rate of change, it is often difficult to determine the exact start time of the reactions. In most cases the performance of the assay, and therefore the reliability of the results, is dependent on the ability of the test system to evenly deliver a certain amount of liquid (usually blood plasma or serum) to a final reactant material. This material must absorb a known quantity of liquid with extreme accuracy and reproducibility in order for the results to be useful. The precise volume measurements required to obtain accurate results with these types of assays present particular challenges and make them difficult to work with.
Both liquid and dry chemistry systems are limited in the concentrations of reactants that can be used. These concentration limits are often due to the presence of highly colored reactants that absorb or reflect light at wavelengths that interfere with or obscure the detection of reaction product which may absorb or reflect light at similar wavelengths. Various methods have been employed in attempts to solve this problem. Hochstrasser, U.S. Pat. No. 3,964,871, describe a disposable indicator for measuring substances which registers the concentration of a substance in a given biological fluid with indicia which are directly readable in a convenient notation thereby reducing reliance on comparison with a color intensity scale. Kim et al., U.S. Pat. No. 4,303,408, describe elements with interferent-reducing zones which remove interferents prior to the reaction zone. Despite these attempts, only marginal improvements are possible due to the physical limitations which are inherent in the methods.
U.S. Pat. No. 5,716,852, issued to Yager et al., teaches a channel-cell system for detecting the presence and/or measuring the presence of analyte particles in a sample stream comprising a laminar flow channel, two inlets in fluid connection with the laminar flow channel for respectively conducting into the laminar flow channel an indicator stream which may comprise an indicator substance which indicates the presence of said analyte particles by a detectable change in property when contacted with said analyte particles, and the sample stream. The laminar flow channel has a depth sufficiently small to allow laminar flow of streams and a length sufficient to allow particles of the analyte to diffuse into the indicator stream to the substantial exclusion of the larger particles in the sample stream to form a detection area. An outlet conducts the streams out of the laminar flow channel to form a single mixed stream. Yager discloses the formation of a stable reaction interface that forms between two fluids moving through a capillary tube in the same direction. We disclose the invention of a stable interface that forms when two liquids meet and stop in a flow matrix after conveying from opposite directions. The Yager patent is predicated on the principle of liquid laminar flow, which was known in the art. In contrast, the present invention employs bibulous material to physically contain the liquid interface.
U.S. Pat. No. 5,187,100, issued to Matzinger et al., discusses a control solution for use with a porous reagent strip, and comprises a flexible semisolid polymer dispersed in water, such as polyvinyl acetate in distilled water, with appropriate control glucose concentration levels. This solution is useful in mimicking whole blood in conjunction with porous reagent strips to determine compliance of the strips and meters to established measurement and performance criteria.
U.S. Pat. No. 5,147,606, issued to Charlton et al., teaches a diagnostic device that detects blood analytes with a sample volume as low as 2 microliters in the hematocrit range of 0% to 60%, or higher. This is accomplished by employing a housing with various chambers and compartments for processing the blood. A sample application port in the housing is used to introduce blood into a metering chamber. From the metering chamber, the blood flows to a reaction chamber for analyzing blood analytes. Blood entering the metering chamber flows into a fluid capillary which indicates that an adequate amount of blood has been received in the metering chamber. The reaction compartment includes a reagent and a filter, the latter of which is disposed between the metering chamber and the reagent so that the reagent reacts with the filtered blood.
U.S. Pat. No. 4,839,297, issued to Freitag et al., teaches a test apparatus for the analytical determination of a component of a body fluid with a base layer and at least two planar test layers which, in the initial state of the test carrier, before carrying out the determination, are separate from one another but can be brought into contact with one another by external manipulation. A first test layer and a second test layer are arranged on the base layer essentially next to one another but separated in the initial state by a gap, a contact element being provided which consists of a capillary-active material which is so dimensioned that it can bridge the gap and which is so mounted and arranged that, in a first position, it cannot contact at least one of the test layers but, by external pressure, it can be brought into a second position in which it contacts both test layers in such a manner that a liquid exchange between the test layers is possible.
U.S. Pat. No. 4,637,978, issued to Dappen, discloses an assay useful for the determination of an analyte in whole blood. In particular, this assay is useful for the quantitative determination of peroxide-generating analytes, such as glucose or cholesterol, in whole blood. This assay utilizes a multizone element consisting essentially of a support having thereon, in order and in fluid contact, a registration zone and a reagent/spreading zone. The reagent/spreading zone has a void volume and average pore size effective to accommodate whole blood, and contains an interactive composition necessary for the analysis. Such composition is capable of providing, upon interaction with the analyte, a dye which can be spectrophotometrically detected at a wavelength greater than about 600 mm.
U.S. Pat. No. 4,160,008, issued to Fenocketti et al., teaches a test device for determining the presence of a liquid sample constituent. The device comprises a base support member having attached to it an indicator member which produces a detectable response, such as a color change, in the presence of the sample constituent. The indicator member comprises an upper reagent layer, a lower absorbent layer and a substantially sample-impervious barrier layer between the upper and lower layers. The indicator member is attached to the base member along the lower side of the absorbent layer.
U.S. Pat. No. 4,042,335, issued to Clement, discloses a multilayer element for the analysis of liquids such as biochemical and biological liquids. The invention includes a reagent layer including a composition that is interactive in the presence of a predetermined substance to be analyzed (analyte) to provide a diffusible, detectable species, e.g., a dye, can be detected. Preferably between the reagent layer and the registration layer, there can be a radiation-blocking layer, such as an opaque reflecting layer, to enhance detection of the diffusible species within the registration layer. A spreading layer is separated from the registration layer by a reagent layer. In operation, a sample of liquid under analysis is applied to the reagent layer or, if present, to a spreading layer. If the sample contains analyte, a chemical reaction or other interaction within the reagent layer provides a detectable species that diffuses, via any intervening layers such as a radiation-blocking layer, into the registration layer for detection there, such as by radiometric techniques like reflection spectrophotometry.
U.S. Pat. No. 3,942,158, issued to Przybylowicz et al., discloses an integral analytical element capable of use in the analysis of liquids, the element having at least two superposed layers including a spreading layer and a reagent layer, in fluid contact. The spreading layer, which can be an isotropically porous layer, spreads within itself at least a component of a liquid sample applied to the element, or a reaction product of such component, to obtain a uniform concentration of at least one such spread substance at the surface of the spreading layer which faces the reagent layer. The reagent layer, which is desirable uniformly permeable to at least one dissolved or dispersed component of the liquid sample or a reaction product of such a component, can include a matrix in which is distributed a material that can interact with, for example, an analyte or analyte reaction product to produce a detectable change in the element, such as one detectable by measurement of electromagnetic radiation. In a preferred embodiment, the interactive material can chemically react with an analyte or analyte reaction product to produce a color change in the element. In another preferred embodiment, the sample spreading layer can filter out chemically interfering or other undesirable materials and obtain selective spreading of sample components and/or it can provide a reflective background, often useful in obtaining analytical results.
U.S. Pat. No. 3,811,840, issued to Bauer et al., teaches a test device for detecting low concentrations of substances in test fluids which includes an absorbent wick having a substantially flat surface portion enclosed in a fluid impervious sheath having an aperture of predetermined limited area formed therein. The aperture is contiguous to and exposes a predetermined limited area of the flat surface portion of the wick, which is incorporated with a reagent specifically reactable with the substance being detected. In use the device is immersed into the test fluid so that the aperture is submerged and the device is allowed to remain therein while the test fluid contacts the reagent area adjacent to the aperture and migrates into the remainder of the wick. The reagent is immobilized with respect to the liquid.
U.S. Pat. No. 5,802,842, issued to Lange et al., discloses a test strip for the detecting of components in liquids, especially in body fluids. The test strip includes a holder and at least one indicator layer containing detection reagents. One surface of the indicator layer is attached to the holder and the other surface is covered with a fine meshwork.
Note, however, that the analytical devices of the above prior art employ fluid movement in only a single direction. Because no reaction interface is created by a movement of two fluids in opposite directions, the above prior art references cannot be employed to measure the reaction intensity or reaction rate at a reaction interface, as disclosed by the present invention.
The present invention provides a solution to the problems and deficiencies of current systems discussed above. Specifically, the present invention provides a device which contain all reactants necessary for sample preparation and analyte detection and methods of their use. The present invention provides devices and methods which eliminate the extreme precision in volume measurement which is required by some methods. The results of assays conducted with the present invention are read in a generic reading area of the device, and a wide variety and versatility in reagent chemistry and concentrations is offered.
The present invention provides a device for detecting and quantifying analytes in a fluid sample. The device is a bibulous material having an application site for the fluid sample in a fluid sample zone and a separate application site for a diluent solution in a diluent solution zone. At least one reagent which is capable of being reconstituted may be present in one or more diluent solution zones and fluid sample zones on the bibulous material. The reagent(s), diluent solution, and fluid sample form a detectable product at a localized interface on the bibulous material upon migration of the fluid sample from one direction and the diluent solution from a second, opposite direction. One or more reagents may be contained on the bibulous material in one or more reagent zones. Reagents to be reconstituted by the fluid sample may be contained by the bibulous material at a site which is closer to the application site for the fluid sample than to the application site for the diluent solution. Conversely, reagents which are to be reconstituted by the diluent solution may be contained by the bibulous material at a site which is closer to the application site for the diluent solution than to the application site for the fluid sample. In a preferred embodiment, the bibulous material may be capable of separating red blood cells from whole blood as the fluid sample travels through the bibulous material. In various embodiments the bibulous material may be a HEMASEP L(copyright) membrane, a HEMASEP V(copyright) membrane, a CYTOSEP(copyright) membrane, a SUPOR(copyright) membrane, or a nitrocellulose membrane. In a preferred embodiment, the detectable reaction product may reflect light. The concentration of unreacted reagents on the bibulous material may provide a reference value, control, or blank for the assay. In other embodiments, the reagent or reagents may be contained in an absorbent pad placed in contact with the bibulous material. The absorbent pad may be selected from the group consisting of cellulose, glass fiber, polyester, or any absorbent polymer.
The present invention also provides methods for detecting and quantifying analytes in a fluid sample. The methods consist of the steps of providing a bibulous material as described above, contacting the fluid sample to be assayed and a diluent solution with the bibulous material at their respective application sites, allowing the fluid sample and the diluent solution to flow toward one another wherein the diluent solution and/or the fluid sample reconstitutes one or more reagents which may be present in the bibulous material, allowing the fluid sample and the diluent solution to converge to form an interface thereby reacting to form a detectable product, and examining the interface and thereby determining the presence or quantity of the analytes in the fluid sample by evaluating the presence or amount of the detectable product. The reagent may be a plurality of reagents in a plurality of locations on the bibulous material. The fluid sample may be whole blood, blood plasma, blood serum, urine, or any body fluid.
The present invention also provides a method for detecting and quantifying analytes in a fluid sample wherein the reagents are added to either or both of the fluid sample and diluent solution prior to contact with the bibulous material. In this aspect, the method will include providing a bibulous material as described above, and the bibulous material may contain no reagents. The methods includes the step of contacting the fluid sample and diluent solution with the bibulous material at their respective application sites, allowing the fluid sample and diluent solution to converge to form an interface thereby reacting to form a detectable product, and examining the interface to determine the presence or quantity of analyte in the fluid sample by evaluating the presence or quantity of the detectable product.